The invention relates to a family of glutamate receptors (GluR) in plants, compounds that modulate the activity of the plant GluR, and the use of such compounds as plant growth regulators, including herbicides. The invention also relates to nucleotide sequences encoding the plant GluR and to plant assay systems designed to identify novel plant growth regulators that may be used as herbicides and/or pharmaceutical drugs.
Glutamate has important roles in plant nitrogen metabolism. Glutamate is the amino acid into which inorganic nitrogen is first assimilated into organic form. Plants have three distinct nitrogen processes related to nitrogen metabolism: (1) primary nitrogen-assimilation, (2) photorespiration, and (3) nitrogen xe2x80x9crecycling.xe2x80x9d All three processes involve assimilation of ammonia into glutamate and glutamine by the operation of glutamine synthetase (GS) and glutamate synthase (GOGAT). Glutamate and glutamine, being the first products of nitrogen-assimilation, in turn serve as nitrogen donors in the biosynthesis of essentially all amino acids, nucleic acids, and other nitrogen-containing compounds such as chlorophyll (Lea et al., in: Recent Advances in Phytochemistry, edited by Poulton et al., New York and London: Plenum Press, 1988, pp. 157-189).
Glutamate is also a principal xe2x80x9cnitrogen-transportxe2x80x9d compound in plants. It and glutamine are two major amino acids used to transport nitrogen within a plant (Lea and Miflin, in: The Biochemistry of Plants, Vol. 5, edited by Stumpf and Conn, Academic Press, 1980, pp. 569-607; Urquhart and Joy, 1981, Plant Physiol. 68:750-754). In light-grown metabolically active plants, glutamate and glutamine are used in anabolic reactions and are transported as such. By contrast, in etiolated or dark-adapted plants, glutamine is converted into inert asparagine for long-term nitrogen storage.
Glutamate also may be a signal or regulatory molecule in regulating the expression of plant genes. Specifically, glutamate along with glutamine and asparagine appears to have an antagonistic role to that of sucrose in regulating certain nitrogen assimilation genes. Sucrose has been shown to induce the expression of genes for nitrate reductase (NR), nitrite reductase (NiR), and chloroplastic glutamine synthetase (GS2) in tobacco (Saur et al., 1987, Z. Naturforsch. 42:270-278; Vincentz et al., 1993, Plant J. 3:315-324). Sucrose also induces genes for GS2 and ferroredoxin-dependent glutamate synthase (Fd-GOGAT) in Arabidopsis. Sucrose-induction of the NR and NiR in tobacco is suppressed by subsequent additions of glutamine, glutamate or asparagine to the media (Vincentz et al., ibid.). Conversely, a nitrogen metabolism gene, glutamine-dependent asparagine synthetase (ASN1), in Arabidopsis is repressed by light or sucrose (Lam et al., 1994, Plant Physiol. 106:1347-1357). The sucrose repression of ASN1 can be relieved by additions of glutamine, glutamate, or asparagine (Id.).
Excitatory amino acids constitute the principal neurotransmitter receptors that mediate synaptic communication in animals (Gasic et al., 1992, Annu. Rev. Physiol. 54: 507-536). In particular, L-glutamate is the major excitatory neurotransmitter of the mammalian central nervous system (Monaghan et al., 1989, Annu. Rev. Pharmacol. Toxic. 29: 365-402). Glutamate signaling in animals is important for many physiological and pathological processes such as developmental plasticity, long-term potentiation, and excitotoxic damage in ischemia and other neurodegenerative disorders (Choi, 1988, Neuron 1: 623-624; Kennedy, 1989, Cell 59: 777-787).
In animals, glutamate can trigger various downstream physiological responses by interacting with different GluR. GluRs in animals are involved in central nervous system (CNS) disorders such as Huntington""s disease, Parkinson""s disease and Alzheimer""s disease. The GluR is involved in the initiation and propagation of seizures and in massive neuronal cell death during periods of ischemia and hypoglycemia. GluRs have been grouped into five distinct subtypes (Gasic et al., 1992, Annu. Rev. Physiol. 54: 507-536): (a) NMDA (N-methyl-D-aspartate), (b) KA (Kainate), (c) AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate), (d) L-AP4 (2-amino-4-phosphonobutyrate) and (e) ACPD (trans-1-amino-cyclopentane-1,3 dicarboxylate). NMPA, KA and AMPA, which form ligand gated ion channels that are activated on a msec scale, are the ionotropic (iGluR) subtypes. By contrast, metabotropic (mGluR) subtypes, L-AP4 and ACPD, are coupled to G proteins and operate on a time scale of several hundred msec to seconds. LAP-4 receptor probably acts via a G protein by increasing the hydrolysis of cGMP and subsequently leads to the closure of ion channels conducting an inward current. The ACPD subtype, which couples with a G protein that is linked to inositol phosphate/diacylglycerol formation and subsequent release of calcium from internal stores. Both iGluR and mGluR seem to play a role in the activation of transcription factors, such as c-jun and c-fos (Condorelli et al., 1993, J. Neurochem. 60: 877-885; Condorelli et al., 1994, Neurochem. Res. 19: 489-499).
There are major differences in the neurophysiological functions of the three subtypes of iGluR (Seeburg, 1995, TINS 16:359-365). AMPA receptors are found in the majority of all fast excitatory neurotransmission. The very low Ca++ permeability of AMPA receptor suggests that they probably do not trigger biochemical reactions directly via an increase in intracellular Ca++ levels. In NMDA receptor, Ca++ flux will trigger different processes ranging from trophic developmental actions to an activity-dependent resetting of the synaptic strength underlying some forms of learning and memory. The significance of high-affinity kainate sites in the nervous systems is yet to be fully understood.
(A) AMPA Receptor
AMPA receptors consist of at least four different subunits: GluR1-GluR4. The two major forms, named xe2x80x9cflipxe2x80x9d and xe2x80x9cflopxe2x80x9d, which are formed by differential splicing, display different expression profiles in the mature and the developing brain (Sommer et al., 1990, Science 249:1580-1585). For GluR2 subunit, RNA editing (Q to R) in transmembrane domain (TM) II has been shown to regulate the Ca++ permeability. RNA editing leads to a decrease in Ca++ permeability (Burnashev et al., 1992, Neuron, 8:189-198; Hume et al., 1991, Science 253:1028-1031).
(B) Kainate Receptors
High-affinity kainate receptors are composed of subunits GluR5-GluR7, KA1, and KA2 (Seeburg et al., 1995, TINS 16:359-365). Both GluR5 and GluR6 subunits also display the Q to R editing similar to the case of GluR2 of AMPA receptors (Sommer et al., 1991, Cell 67:11-19). GluR6 has two additional positions in TMI that are modified by RNA editing (Kohler et al., 1993, Neuron 10:491-500). For GluR6, only when TMI is edited does editing in TMII (Q to R) influence Ca++ permeability (Kohler et al., 1993, Neuron 10:491-500). In contrast to the AMPA receptor channel, GluR6(R) channels edited in TMI show a higher Ca++ permeability than GluR6(Q) channels (Kohler et al., 1993, Neuron 10:491-500).
(C) NMDA Receptor
NMDA receptors are highly permeable to Ca++. The NMDA receptor can be reconstituted as heteromeric structures from two subunit types: NRI and one of the four NR2 (NR2A-NR2D) (Seeburg, 1995, TINS 16:359-365). All of the subunits do not show RNA editing in TMI and TMII. In fact all subunits contain an N at the site which Q to R editing occurs in non-NMDA iGluR. The most distinct feature of NMDA receptors is that they require both glycine and glutamate or both glycine and NMDA to activate the channel. The NMDA receptor has been linked to regulation of coccidian rhythm in rat brains.
In contrast to ionotropic glutamate receptors. (iGluR) the hallmark of the mGluR receptors resides on the fact that these molecules are coupled to G proteins and thus able to elicit typical G protein-driven intracellular responses (Gasic et al., 1992, Annu. Rev. Physiol. 54:507-536; Minakami et al., 1994, Biochem. Biophys.. Res. Commun. 199:1136-1143; Schoepp et al., 1993, Trends in Pharmacol. Sci. 14:13-20).
The cloning of mGluR1 from a expression cDNA library of a rat cerebellum (mGluR1a), was followed by the cloning and characterization of six other mGluR genes (Schoepp et al., 1993, Trends in Pharmacol. Sci. 14:13-20). The mGluR1 a, the prototype member of the family, possess a large extracellular domain, a putative xe2x80x9cseven passxe2x80x9d transmembrane region and display highly conserved amino acids with other members of the mGluRs both at the membrane spanning region, extracellular region and intracytoplasmic loops between transmembrane domains (Gasic et al., 1992, Annu. Rev. Physiol. 54:507-536; Minakami et al., 1994, Biochem. Biophys. Res. Commun. 199:1136-1143; Schoepp et al., 1993, Trends in Pharmacol. Sci. 14:13-20). The mGluR genes are unique in that they do not show significant homology with any of the previously characterized G proteins (Nakanishi et al., 1994, In: Toward a molecular basis of alcohol use and abuse. ed. by Jansson et al. p 71-80; Schoepp et al., 1993, Trends in Pharmacol. Sci. 14:13-20) and very little is known on the signal transduction mechanisms and second messenger responses for each mGluR receptor (Schoepp et al., 1993, Trends in Pharmac. Sci. 14:13-20).
Studies in the in situ localization of mRNA encoding the different mGluRs shows them to be differentially distributed in the brain with cells from diverse tissues expressing one or more combinations of the various members of the mGluR family of receptors, suggestive of a relevant participation in the modulation of several important biological processes (Schoepp et al., 1993, Trends in Pharmac. Sci. 14:13-20). Indeed, the mGluR proteins have been reported to be involved with neuroprotection and neuronal pathophysiology (Baskys, 1992, Trends in Neuro Sci. 15:92-96; Schoepp et al., 1993, Trends in Pharmac. Sci. 14:13-20).
Analysis of the pharmacological properties of the individual mGluR molecules revealed that the agonists L-AP4 (L-2-amino-4-phosphonobutyrate) and ACPD (trans-1-amino-cyclopentane-1,3 dicarboxylate) can selectively stimulate different mGluR serving as a basis for the classification of this group of proteins (Nakanishi et al., 1994, In: Toward a molecular basis of alcohol use and abuse. Ed. Jansson et al. p 71-80).
(A) L-AP4 Receptor
The L-AP4 receptor has been defined electrophisologically as an inhibitory glutamate site and biochemical evidence suggest that mGluR4, mGluR6 and mGluR7 are involved in this response (Nakanishi et al., 1994, In: Toward a molecular basis of alcohol use and abuse ed by Jansson et al. p 71-80; Schoepp et al., 1993, Trends in Pharmacol. Sci. 14:13-20). The L-AP4 receptor appears to be localized pre-synaptically such that activation inhibits the release of excitatory neurotransmitter through a mechanism involving a pertussis toxin sensitive G protein (Nakanishi et al., 1994, In: Toward a molecular basis of alcohol use and abuse. Ed. Jansson et al. p 71-80). The molecular identity and possible biological function of this group of receptors come from studies where mammalian cells were transfected with a cloned isoform of mGluR (mGluR4) responded to both L-glutamate and L-AP4 by depressing forskolin-stimulated cAMP levels (Nakanishi et al., 1994, In: Toward a molecular basis of alcohol use and abuse. ed. by Jansson, p 71-80; Schoepp et al., 1993, Trends in Pharmacol. Sci. 14:13-20). L-AP-4 has also been shown to reduce electrically-stimulated excitatory transmission, suggestive of a close interaction between mGluR and Ca2+ channels (Cunningham et al., 1993, Life Sciences 54:135-148; Schoepp et al., 1993, Trends in Pharmac. Sci. 14:13-20) and specifically in the regulation of ionotropic glutamate receptors (Baskys, 1992, Trends in Neuro. Sci. 15:92-96). Very little is known about the signal transduction mechanisms and second messenger responses elicited by this subgroup of mGluR.
(B) ACPD Receptor
The mechanisms of signal transduction of this subgroup of receptors is better understood than that of the L-AP4-responsive mGluRs. Transfection of the cDNA for mGluR1, mGluR2, mGluR3 and mGluR5 in CHO cells revealed that this receptors are strongly responsive to the drug ACPD (trans-1-amino-cyclopentane-1,3 dicarboxylate) (Cunningham et al., 1993, Life Sciences 54:135-148; Schoepp et al., 1993, Trends in Pharmac. Sci. 14:13-20). However, not all mGluR activate the same pathways and different mGluR can elicit diverse intracellular responses. Thus, mGluR5 possess high homology with mGluR1 yet these two receptors differ in which mGluR5 does not induce formation of cAMP. Moreover, stimulation of the mGluR2 and 3 does not lead to phosphoinositide hydrolysis and mGluR2 has been shown to inhibit cAMP formation in transfection experiments (Schoepp et al. 1993). The diversity of the mGluR family of receptors can be further appreciated by the recent observation that mGluR2 and mGluR3 receptors display an unusually high response to stimulation by quisqualate when compared to other ACPD mGluR responses (Nakanishi et al., 1994, In: Toward a molecular basis of alcohol use and abuse. ed. by Jansson et al. p 71-80; Schoepp et al., 1993, Trends in Pharmacol. Sci. 14:13-20) which argues in favor of their further grouping in a more specialized division among the ACPD-induced receptors. Finally, stimulation of primary neuronal cultures with ACPD and quisqualate caused a strong and transient induction of immediate early genes such as c-fos, c-jun and zif-268 mRNAs (Condorelli et al., 1994, Neurochem Res. 19:489-499).
The present invention relates to a family of GluR in plants, including ionotropic (iGluR), metabotropic (mGluR) and other glutamate-like plant receptors. The plant GluRs of the invention may function as signal transducers involved in the regulation of plant growth. The invention also relates to the identification of compounds that modulate the activity of the plant GluR, and the use of such compounds as plant growth regulators, including herbicides.
The invention is based in part, on a number of unanticipated surprising discoveries. One is the discovery of plant proteins that have high degree of amino acid sequence homology to the animal ionotropic or metabotropic glutamate receptors previously found only in vertebrate tissues. The other is the finding that agonists and antagonists of animal glutamate receptors function to modulate expression of plant genes and as plant growth regulators. These agonists and antagonists structurally constrained or do not resemble glutamate. Thus, their actions in plants likely are due to their specific interaction with one or more plant glutamate receptors, rather than to general effects on glutamate-utilizing enzymes. These findings together indicate that plants have glutamate receptors that function as signal transducers.
The invention encompasses: (a) nucleotide sequences that encode the plant GluR, including mutants, recombinants, and fusion proteins; (b) the expression of such nucleotide sequences in genetically engineered host cells and/or in transgenic plants; (c) the isolated GluR plant proteins and GluR engineered gene products, including mutants, fragments, and fusion proteins; (d) antibodies to the plant GluR proteins and polypeptides; (e) screening assays involving the use of plants, transgenic plants, genetically engineered cells that express the plant GluR or mutants thereof, or GluR proteins or peptides, to identify compounds that act as agonists or antagonists; (f) the use of such agonists or antagonists as plant growth regulators, including herbicides; (g) the engineering of transgenic plants resistant to herbicidal antagonists of the plant GluR and/or transgenic plants with improved agronomic or industrial properties; and (h) the use of antagonists or agonists of the plant GluR identified in the screening assays described herein as drugs for animal use, including humans.
An agonist is defined herein as an agent that acts like a referenced compound or that activates a receptor molecule.
An antagonist is defined herein as an agent that acts in opposition to an agonist or a referenced compound or that inhibits a receptor molecule.
A chimeric gene comprises a coding sequence linked to a regulatory region, i.e., promoters, enhancer elements and additional elements known to those skilled in the art that drive and regulate expression, that said coding sequence is not naturally linked to. The coding sequence may encode messenger RNA (mRNA), antisense RNA or ribozymes.
FIGS. 1A,1B,1C. HPLC Analysis of Free Amino Acids in Arabidopsis
FIG. 1A. Amino acids were extracted from leaves of Arabidopsis plants that were grown in light (empty boxes) or subsequently dark adapted for 24 hours (filled boxes). Amino acids were derivatized and separated by reverse phase HPLC. Each sample represents the average of three different plants (two leaves/plant). The standard three letter code is used for all amino acids; gaba: xcex3-amino burytic acid.
FIG. 1B. Average amino acid content in phloem exudates of three independent plants (one leaf/plant).
FIG. 1C. Average amino acid content of xylem sap collected from cut hypocotyls of three independent plants. Data are from Schultz (1994).
FIG. 2. Reciprocal Control By Light On Arabidopsis GLN2 and ASN1 Expression
The effects of light on GLN2 and ASN1 expression were tested in mature Arabidopsis plants. Plants were grown on soil under a 16-h light/8-h dark cycle for 2 weeks and transferred to continuous light (lane 1) or continuous darkness (lane 2) for 5 d. Total RNA (10 xcexcg) was used for each of the lanes. Hybridization was performed by [xcex1-32P]dATP-labeled GLN2 or digoxigenin-labeled ASN1 DNA probes in Strategene QuikHyb solution under high stringency condition. The nylon filter was first hybridized with the GLN2 probe, then stripped, and rehybridized with the ASN1 probe. (Lam et al., 1994).
FIG. 3. Effect Of C:N Ratio On The mRNA Levels Of ASN1 And GDH
Arabidopsis seeds were grown on plates containing MS medium plus 3% (w/v) Suc under 16-h light/8-h dark cycle for 2 weeks. The plants were then transferred to media described below and grown in complete darkness for 2.5 d. Lanes 1 to 4, MS medium with no sugar; lanes 5 to 8, MS medium with 3% (w/v) Suc. MS was supplemented with 0.4 mM Asn (lanes 2 and 6), 3.4 mM Gln (lanes 3 and 7), or 3.3 mM Glu (lanes 4 and 8). The expression of ASN1, GDH, and a cytosolic GS (GSR2) were detected by northern analyses (under high-stringency conditions in 50% [v/v] formamide solution). 10 xcexcg of total RNA was used for each lane. The nylon filter was first hybridized with ASN1, then stripped, and re-hybridized with the GSR2 probe. The nylon filter was then stripped again and re-hybridized with the GDH probe.
FIGS. 4A and 4B. A Model Depicting The Regulation Of Nitrogen Assimilation Genes By C:N Ratio
FIG. 4A. In the light, when photosynthesis occurs and carbon skeletons are abundant, nitrogen is assimilated and transported as glutamine and glutamate; levels of mRNA for genes involved in glutamine and glutamate synthesis (GLN2, GLU1) are accordingly induced by both light and sucrose. By contrast, light represses the synthesis of asparagine which therefore accumulates only in tissues of dark-adapted plants.
FIG. 4B. Levels of ASN1 mRNA are dramatically induced in dark-adapted plants, and this induction is repressed by light or by high levels of sucrose. Thus, under conditions of carbon limitation or nitrogen excess, plants activate genes for asparagine biosynthesis (Lam et al., 1995). The mRNA level of GDH was found to be under similar control (see also FIG. 3).
FIG. 5. Proposed Topology and Functional Domains of Ionotropic Glutamate Receptor Subunits
Hydrophobicity plots of GluR subunit sequences predict four transmembrane (TM) segments (TM I-IV), depicted here hypothetically as a helices I-IV.
FIG. 6. Peptide Sequence Homology Between The Arabidopsis iGluR (SEQ ID NO1) and Animal iGluRs [E. coli GlnH:(SEQ ID NO:2); Chick KBP:(SEQ ID NO:3); Frog KBP;(SEQ ID NO:4); Rat GluR-K1: (SEQ ID NO:5); Rat GluR-K3:(SEQ ID NO:6); Rat GluR-K2:(SEQ ID NO:7)]
Peptide sequence analysis shows that the putative Arabidopsis iGluR contains a conserved glutamine binding domain which exists in all animal iGluRs.
FIG. 7A. Peptide sequence analysis shows the extensive homology between the putative Arabidopsis iGluR (SEQ ID NO:8) animal iGluRs. The region of homology extends from the glutamine binding domain into the transmembrane domains.
FIG. 7B. Peptide sequence analysis shows the extensive homology between the putative Arabidopsis mGluR and animal iGluR [NMDA:(SEQ ID NO:9); KA:(SEQ ID NO:10)]. The region of homology extends from the glutamate binding domain into the transmembrane domain.
FIG. 7C. Arabidopsis EST clones with low degree homology to glutamate binding domains. These EST clones have no homology to ionotropic nor metabotrobic GluR. Partial nucleotide sequence of EST clones are provided here [ATT50711:(SEQ ID NO:13); ATT52655:(SEQ ID NO:14; T20773:(SEQ ID NO:15)].
FIG. 8. Genomic Southern Analysis of Arabidopsis iGluR
Two xcexcg of CsCl-purified Arabidopsis genomic DNA was digested with different restriction enzymes. Genomic Southern blot analyses were performed by running the digested DNA on a 1% (w/v) Tris-phosphate-EDTA agarose gel. The DNA was transferred to a nylon membrane after depurination, denaturation, and neutralization steps, followed by high-stringency hybridization with DIG-labeled probes which are generated by random-primed reactions (as described in the Boehringer-Mannheim Genius System User""s Guide).
FIG. 9. Expression Of Arabidopsis iGluR In Different Tissues
Twenty xcexcg of total RNA from each of the leaf, root, and flower tissues were run on a 1% formaldehyde agarose gel. Northern blot analyses were performed with high-stringency hybridization conditions at a temperature of 42xc2x0 C. in 50% (v/v) formamide hybridization solution. Washing and chemiluminescent detection were performed according to the Boehringer-Mannheim Genius System User""s Guide. The Northern shows that Arabidopsis iGluR mRNA is expressed predominantly in leaves and also at lower levels in roots and flowers of Arabidopsis.
FIG. 10. Chemical Structures of Glutamate, Kainate, and DNQX
FIGS. 11A and 11B. Effects Of iGluR Agonist and On The Growth Of Arabidopsis
Arabidopsis seeds were grown on MS+3% sucrose vertical tissue culture plates containing various amounts of kainate (FIG. 11A) or DNQX (FIG. 11B), with (white bars) or without (black bars) glutamate supplementation. The effects of each drug on plant growth were assayed by measuring root length after two week. The results were discussed in text.
FIGS. 12A, 12B, 12C. Induction of Gene Expression by iGluR Agonist
FIG. 12A. Arabidopsis seeds were grown on plates containing MS medium plates 3% (w/v) Suc under 16-h light/8-h dark cycle for 2-3 weeks. The plants were then transferred to media described below and grown in complete darkness for 2 d. All samples containing 3% sucrose except for lane 2. MS was supplemented with kainate (lane 4:0.3 mM and lane 5:0.03 mM), 0.05% (w/v) glutamate (lanes 6 and 7), and 10 xcexcM DNQX (lanes 7 and 8). The expression of ASN1 and a control gene were detected by northern analyses on duplicate blots (under high-stringency conditions in 50% [v/v] formamide-solution), 20 xcexcg of total RNA was used for each lane.
FIG. 12B. Quantitation of the Northern blot results in (A) by densitometry scan.
FIG. 12C. Average folds of induction in two Northern lot experiments.
FIGS. 13A and 13B. Inhibition of Arabidopsis Growth by High Dosage of Kainate and DNQX
Photographic representation of high dosage inhibitory effects of kainate and DNQX on the growth of Arabidopsis as described in FIG. 11.
FIGS. 14A and 14B. Model Depicting Effects of Agonists and Antagonists on Plants Expressing Wildtype and Mutant GluR.
FIG. 15. Nucleotide and deduced amino acid sequence of full length Arabidopsis iGluR cDNA, called iGlr1. The regions of highest homology to animal iGluR are denoted in FIGS. 17 and 18.
The full-length Arabidopsis iGlr1 cDNA clone was constructed as follows: the partial EST cDNA clone 107M14T7 was used as a hybridization probe to isolate two additional iGlr cDNA clones (HM299 and HM262) from two different Arabidopsis cDNA libraries, KC-HM1 and CD4-7 (obtained from the Arabidopsis stock center, Ohio). Portions of each iGlr cDNA clone were annealed to generate a full-length Arabidopsis iGlr1 cDNA which was given the trivial name HM330.
FIG. 16. Proposed membrane topology of iGluR receptors in animals. The model shows the important domains of animal iGluRs and their membrane topology. This figure is included as a reference, and does not include data generated in our lab. In addition to a signature 3+1 transmembrane topology, animal iGluRs contain two extracellular domains which are proposed to bind to glutamate. The two putative glutamate bindings domains have been previously shown to have homology to the E. coli glutamine permease gene (GlnH).
FIG. 17. Conserved domains between animal and Arabidopsis iGluR gene. The Arabidopsis iGlr1 cDNA encodes numerous conserved features of animal iGluRs including, 1. a signal peptide to direct it to the membrane (SP), 2. two putative glutamate-binding domains with homology to E. coli (GlnH1 and GlnH2), 3. Four transmembrane domains (TM I-IV). The amino acid sequences spanning the high homology region are shown in FIG. 4.
FIG. 18. Amino acid identities between Arabidopsis iGlr1 (SEQ ID NOS:26-27) and iGluR (SEQ ID NOS:28-31) gene of rat. Boxed are the GlnH1 and GlnH2 domains which show homology to E. coli glutamine permease (as defined in the animal sequence; see FIG. 17), the four transmembrane domains (TM I-IV). The arrow points to the conserved ligand-binding residue in the GlnH1 domain.
FIG. 19. A proposed role for plant iGluR in light signal transduction. We have shown that the iGluR antagonist DNQX and high concentrations of the iGluR agonist KA can block photomorphogenic processes such as germination, chloroplast development and hypocotyl elongation (see FIGS. 20-23). This model proposes a role for plant iGluR in the light signal transduction cascade.
FIG. 20. iGluR antagonist DNQX phenocopies Arabidopsis long hypocotyl (hy) mutants impaired in light signal transduction. Light normally promotes greening and inhibits hypocotyl elongation in wild-type seedlings. hy mutants are impaired in light perception/signal transduction. hy mutants when grown in light take on the morphology of dark-grown seedlings (long hypocotyl). When wild-type plants are treated with DNQX, they grow as hy mutants (long hypocotyl).
FIG. 21. DNQX has a significant effect on hypocotyl length in Arabidopsis. Increasing doses of DNQX (200 uM and 400 uM) cause significant increases in hypocotyl elongation in Arabidopsis. N=number of plants measured.
FIG. 22. DNQX, the iGluR antagonist blocks light-induced chloroplast development in Arabidopsis. Left panel. Plants grown in darkness have unopened yellow cotyledons. When these plants are exposed to light for 5 hrs, the cotyledons begin to green. If plants are grown in the dark with DNQX in the media, the cotyledons remain yellow and unopened after 5 hrs of light exposure. Thus, DNQX appears to block light-induced chloroplast development.
FIG. 23. Effects of kainate of germination of Arabidopsis in the dark. Arabidopsis seedlings germinated on media containing increasing amounts of kainate (200-400 uM) show a significant inhibition of germination of dark-grown seedlings. This inhibition of germination is likely to be specific to iGluR as it is specifically reversed by the supplementation of glutamate to the growth media.
FIG. 24. The Arabidopsis iGlr1 gene was mapped using recombinant inbred lines of Arabidopsis. An RFLP for iGlr1 was identified in the wild-type Arabidopsis ecotypes Columbia (C) and Landsberg (L). This iGlr1-specific RFLP was used to identify the genotype of the iGlr1 gene in 30 Recombinant Inbred lines as being derived from the C or L parents. The xe2x80x9cpatternxe2x80x9d of inheritance of the iGlr1 gene in the recombinant inbred lines was compared to known markers and used to determine a map position (see FIG. 25).
FIG. 25. iGlr1 maps to chromosome III to a similar position as two known mutants, hy2 and spy. Using data from recombinant inbred lines hy2 is a mutant impaired in light signal transduction (see review Whitelam and Harberd, Plant Cell Environment 1994, 17, 615-625). The spy mutant is impaired in GA hormone signal transduction (Jacobsen and Olszewski, 1993, Plant Cell 5, 887-896).
FIG. 26. Screen for Arabidopsis mutants with altered sensitivity to the iGluR antagonist DNQX. Mutants affected in iGluR can be used to test the in vivo function of plant iGluR and could also be mapped relative to the cloned gene. To isolate mutants in Arabidopsis iGluR we developed a screen for plants that are super-sensitive to the iGluR antagonist DNQX. Normally wild-type Arabidopsis only show an elongated hypocotyl phenotype when exposed to high doses of DNQX (200-400 xcexcM) and show no hypocotyl elongation at low-concentrations (100 xcexcM) (see FIG. 21). Therefore, ems mutagenized M2 seeds were germinated on media containing 100 uM DNQX and look for mutants that displayed an elongated hypocotyl at this low dose of DNQX.
FIGS. 27A-D. Isolation of putative Arabidopsis mutants that are super-sensitive to the iGluR antagonist DNQX. Arabidopsis wild-type seedlings show no significant hypocotyl elongation when germinated on 100 uM DNQX (FIG. 27B) compared to control MS (FIG. 27A). By contrast, the putative DNQX supersensitive mutant shows an elongated hypocotyl when germinated on 100 uM DNQX (FIG. 27D). The putative super-sensitive mutant also shows an elongated hypocotyl compared to wild-type when germinated in the absence of DNQX (FIG. 27C). This is not unexpected that a mutation in iGluR would cause a phenotype of light-insensitivity.
FIGS. 28A and 28B. Selection of Arabidopsis mutants resistant to the iGluR agonist Kainate. High doses of kainate (12 mM) kill wild-type seedlings. This is not unexpected as high doses of the iGluR agonist kainate function as a neurotoxin in animals. Arabidopsis mutants with putative defects in the KA binding site of iGluR were selected for the ability to grow in the presence of 12 mM kainate. FIG. 28A shows ems mutagenized Arabidopsis M2 seedlings sown on 12 mM kainate. Note the one KA-resistant plant is enlarged in FIG. 28B.
FIG. 29. Arabidopsis mutants resistant to the iGluR agonist kainate, display a Giant phenotype. Three independent Arabidopsis were mutants selected for growth on 12 mM KA (see FIG. 28). When the putative KA-resistant plants are transferred to soil, they each display varying degrees of a Giant vegetative phenotype. This result may indicate that iGluR affects/enhances overall plant growth.
FIG. 30. Hydropathy plots of plant vs. animal iGluR. The hydropathy plot and prediction of transmembrane regions based on deduced primary sequence of rat iGluR K2 (upper panel) and Arabidopsis iGlr1 (lower panel) were performed with the program TMpred (K. Hofmann and W. Stoffel, TMbasexe2x80x94A database of membrane spanning proteins segments, Biol. Chem. Hoppe-Seyler 347,166 (1993)). Results showed that in both animal and plant iGluRs, there is a N-terminal signal peptide as well as a 3+1 transmembrane domain at the C-terminal region. These are structural features conserved in all iGluR genes.
FIG. 31. Inhibitory effects of iGluR antagonist DNOX on light-induced chlorophyll synthesis in Arabidopsis. Seedlings were sown on MS+3% sucrose agar plates and grown in complete darkness (etiolated) for 5 days. On day 6, half of the seedlings were kept in the dark (D) and the other half were transferred to white light (L) for 7.5 hours. Levels of chlorphyll a and chlorophyll b were measured using the method by Moran (Plant Physiol. 1982, 69:1376-1381). The cotyledons of 30-40 plants were used for each data point. The results of each treatment were an average of three groups of plants. The error bars represent standard deviation. Black bar=no DNQX treatment. White bar=treatment with 0.4 mM DNQX. Treatment with DNQX resulted in a 30-35% reduction in light-induced chlorophyll synthesis.
FIG. 32. Late flowering in KA-resistant xe2x80x9cgiantxe2x80x9d mutants. Two independent KA-resistant mutants (KA-giant-1 and KA-Giant-2) show a giant, late flowering phenotype. Some of the cauline leaves from the flowering stalks of these KA-resistant giant mutants (panel B) display the morphology of rosette leaves in constrast to the normal morphology in wild type plants (panel A). These results suggest that iGluR may be involved in flowering and developmental processes.
FIG. 33. Isolation of a homeotic mutant from the DNQX supersensitive screen. One DNXQ supersentitive mutant (0.1DNQX-10) showed a homeotic phenotype. Lateral roots emerged from the elongation hypocotyl of the plant (shown by the arrows). In wild type plants, lateral roots emerge only from roots.